Connective Tissue Growth Factor (CTGF)
CTGF is a 36 kD, cysteine-rich, heparin binding, secreted glycoprotein originally isolated from the culture media of human umbilical vein endothelial cells. (See e.g., Bradham et al. (1991) J Cell Biol 114:1285-1294; Grotendorst and Bradham, U.S. Pat. No. 5,408,040.) CTGF belongs to the CCN (CTGF, Cyr61, Nov) family of proteins (secreted glycoproteins), which includes the serum-induced immediate early gene product Cyr61, the putative oncogene Nov, the ECM-associated protein FISP-12, the src-inducible gene CEF-10, the Wnt-inducible secreted protein WISP-3, and the anti-proliferative protein HICP/rCOP (Brigstock (1999) Endocr Rev 20:189-206; O'Brian et al. (1990) Mol Cell Biol 10:3569-3577; Joliot et al. (1992) Mol Cell Biol 12:10-21; Ryseck et al. (1990) Cell Growth and Diff 2:225-233; Simmons et al. (1989) Proc Natl Acad Sci USA 86:1178-1182; Pennica et al. (1998) Proc Natl Acad Sci USA, 95:14717-14722; and Zhang et al. (1998) Mol Cell Biol 18:6131-6141.) CCN proteins are characterized by conservation of 38 cysteine residues that constitute over 10% of the total amino acid content and give rise to a modular structure with N- and C-terminal domains. The modular structure of CTGF includes conserved motifs for insulin-like growth factor binding protein (IGF-BP) and von Willebrand's factor (VWC) in the N-terminal domain, and thrombospondin (TSP1) and a cysteine-knot motif in the C-terminal domain.
CTGF expression is induced by members of the Transforming Growth Factor beta (TGFβ) superfamily, which includes TGFβ-1, -2, and -3, bone morphogenetic protein (BMP)-2, and activin, as well as a variety of other regulatory modulators including dexamethasone, thrombin, vascular endothelial growth factor (VEGF), and angiotensin II; and environmental stimuli including hyperglycemia and hypertension. (See, e.g., Franklin (1997) Int J Biochem Cell Biol 29:79-89; Wunderlich (2000) Graefes Arch Clin Exp Opthalmol 238:910-915; Denton and Abraham (2001) Curr Opin Rheumatol 13:505-511; and Riewald (2001) Blood 97:3109-3116; Riser et al. (2000) J Am Soc Nephrol 11:25-38; and International Publication No. WO 00/13706). TGFβ stimulation of CTGF expression is rapid and prolonged, and does not require persistent application. (Igarashi et al. (1993) Mol Biol Cell 4: 637-645.) Enhanced expression of CTGF by TGFβ involves transcriptional activation via DNA regulatory elements present in the CTGF promoter. (Grotendorst et al. (1996) Cell Growth Differ 7: 469-480; Grotendorst and Bradham, U.S. Pat. No. 6,069,006; Holmes et al. (2001) J Biol Chem 276:10594-10601.)
CTGF has been shown to increase steady-state transcription of α1(I) collagen, α5 integrin, and fibronectin mRNAs, as well as to promote cellular processes including proliferation and chemotaxis of various cell types in culture. (See e.g., Frazier et al. (1996) J Invest Dermatol 107:406-411; Shi-wen et al. (2000) Exp Cell Res 259:213-224; Klagsburn (1977) Exp Cell Res 105:99-108; Gupta et al. (2000) Kidney Int 58:1389-1399; Wahab et al. (2001) Biochem J 359(Pt 1):77-87; Uzel et al. (2001) J Periodontol 72:921-931; and Riser and Cortes (2001) Ren Fail 23:459-470.) Subcutaneous injection of CTGF in neonatal mice results in the local deposition of granulation tissue. Similarly, subcutaneous injection of TGFβ generates granulation tissue formation and induces high levels of CTGF mRNA in local fibroblasts. Moreover, combination or sequential treatment with TGFβ and CTGF results in the development of a more persistent granuloma. (Mori et al. (1999) J Cell Physiol 181:153-159.) Thus, CTGF appears to mediate a subset of the effects elicited by TGFβ, in particular, the production and deposition of extracellular matrix (ECM). Further, the ability to respond to CTGF, or the extent of the CTGF response, may rely upon a priming stimulus provided by TGFβ treatment that enables cellular “competence.” (International Publication No. WO 96/08140.)
Although a plethora of interacting factors have been characterized that modulate tissue organization, a consensus is now emerging for the role of CTGF in regulating skeletal development, wound healing and extracellular matrix (ECM) remodeling, fibrosis, tumorigenesis, and angiogenesis. For example, elevated CTGF expression has been observed in cirrhotic liver, pulmonary fibrosis, inflammatory bowel disease, sclerotic skin and keloids, desmoplasia, and atherosclerotic plaques. (Abraham et al. (2000) J Biol Chem 275:15220-15225; Dammeier et al. (1998) Int J Biochem Cell Biol 30:909-922; diMola et al. (1999) Ann Surg 230(1):63-71; Igarashi et al. (1996) J Invest Dermatol 106:729-733; Ito et al. (1998) Kidney Int 53:853-861; Williams et al. (2000) J Hepatol 32:754-761; Clarkson et al. (1999) Curr Opin Nephrol Hypertens 8:543-548; Hinton et al. (2002) Eye 16:422-428; Gupta et al. (2000) Kidney Int 58:1389-1399; Riser et al. (2000) J Am Soc Nephrol 11:25-38.)
CTGF is also upregulated in glomerulonephritis, IgA nephropathy, focal and segmental glomerulosclerosis and diabetic nephropathy. (See, e.g., Riser et al. (2000) J Am Soc Nephrol 11:25-38.) An increase in the number of cells expressing CTGF is also observed at sites of chronic tubulointerstitial damage, and CTGF levels correlate with the degree of damage. (Ito et al. (1998) Kidney Int 53:853-861.) Further, CTGF expression is increased in the glomeruli and tubulointerstium in a variety of renal diseases in association with scarring and sclerosis of renal parenchyma. Elevated levels of CTGF have also been associated with liver fibrosis, myocardial infarction, and pulmonary fibrosis. For example, in patients with idiopathic pulmonary fibrosis (IPF), CTGF is strongly upregulated in biopsies and bronchoalveolar lavage fluid cells. (Ujike et al. (2000) Biochem Biophys Res Commun 277:448-454; Abou-Shady et al. (2000) Liver 20:296-304; Williams et al. (2000) J Hepatol 32:754-761; Ohnishi et al. (1998) J Mol Cell Cardiol 30:2411-22; Lasky et al. (1998) Am J Physiol 275: L365-371; Pan et al. (2001) Eur Respir J 17:1220-1227; and Allen et al. (1999) Am J Respir Cell Mol Biol 21:693-700.) Thus, CTGF represents a valid therapeutic target in disorders, such as those described above.
The association of CTGF with various aspects of these disorders has been established; and methods for treating disorders through modulation of CTGF have been described. (See, e.g., Grotendorst and Bradham, U.S. Pat. No. 5,783,187; International Publication No. WO 00/13706; and International Publication No. WO 03/049773.) Modulation of growth factors, cytokines, and cell surface receptors can be accomplished using monoclonal antibodies, and several therapeutic monoclonal antibodies have been approved or are underdevelopment. (See, e.g., Infliximab (Remicade; Maini et al. (1998) Arthritis Rheum 41:1552-1563; Targan et al. (1997) N Engl J Med 337:1029-1035); Basiliximab (Simulect) and Daclizumab (Zenapax) (Bumgardner et al. (2001) Transplantation 72:839-845; Kovarik et al. (1999) Transplantation 68:1288-1294); and Trastuzumab (Herceptin; Baselga (2001) Ann Oncol 12 Suppl 1:S49-55.))
Antibodies have been generated against CTGF, and have proven efficacious in vivo at, e.g., inhibiting angiogenesis. (See, e.g., Grotendorst and Bradham, U.S. Pat. No. 5,408,040; International Publication No. WO 99/07407; and Shimo et al. (2001) Oncology 61:315-322). Further, the modular nature of CTGF appears to distinguish domains involved in specific biological activities. For example, the N-terminal half of CTGF has been shown to stimulate cell differentiation and ECM production, whereas the C-terminal half stimulates cell proliferation. (See, e.g., International Publication Nos. WO 00/35936 and WO 00/35939; and Brigstock and Harding, U.S. Pat. No. 5,876,70.) This demonstrates that antibodies directed to different regions of the CTGF molecule exhibit different effects with respect to modulating biological activities of CTGF. (See, e.g., International Publication Nos. WO 00/35936 and WO 00/35939). Currently, no clear distinction has been made between anti-CTGF antibodies that produce a desired effect, and those which either produce multiple effects or are non-neutralizing. (See, e.g., International Publication No. WO 99/33878.)
There is clearly a need in the art for agents that effectively neutralize the activity of CTGF in disease. Antibodies, particularly monoclonal antibodies, provide the specificity and pharmacokinetic profiles appropriate for a therapeutic agent, and neutralizing antibodies targeted to specific activities of CTGF would fulfill a need in the art and would find use in therapeutic treatment of CTGF-associated disorders including pulmonary disorders such as idiopathic pulmonary fibrosis (IPF), etc.; renal disorders such as diabetic nephropathy, glomerulosclerosis, etc.; and ocular disorders such as retinopathy, macular degeneration, etc.